Subko Karolina, Wang Xinhui, Nielsen Frederik H, Isbrandt Thomas, Gotfredsen Charlotte H, Ramos Maria C, Mackenzie Thomas, Vicente Francisca, Genilloud Olga, Frisvad Jens C, Larsen Thomas O
Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark.
Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.
Front Microbiol. 2021 Jan 15;11:618730. doi: 10.3389/fmicb.2020.618730. eCollection 2020.
Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of , a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.
曲霉芬amate是一种小肽天然产物，因其抗肿瘤活性而备受关注。近年来，已报道了许多具有生物活性的合成曲霉芬amate类似物，而仅发现了少数微生物或植物来源的天然类似物。在此，我们描述了一种用于发现和设计新型曲霉芬amate类似物的超高效液相色谱-高分辨质谱/质谱（UHPLC-HRMS/MS）和氨基酸补充方法。对曲霉芬amate的丰富生产者进行化学分析，发现了三种先前描述的和两种大量产生的新型曲霉芬amate类似物，这表明通过改变氨基酸可用性有可能生物合成更多的曲霉芬amate类似物。随后在富含蛋白原性和非蛋白原性氨基酸的培养基上生长，发现了一系列新型曲霉芬amate类似物，包括单氨基酸或双氨基酸交换，以及苯甲酸被烟酸取代，后者首次从天然来源观察到。总共通过HRMS/MS对22种新的曲霉芬amate类似物进行了表征，其中一种通过分离和核磁共振结构解析得到了进一步确认。这项研究表明基于底物可用性的非凡非核糖体肽合成酶（NRPS）灵活性，因此在丝状真菌中操纵和设计新型肽天然产物具有潜力。
